Induction of 25-OH-vitamin D3 24- and 23-hydroxylase activities in partially purified renal extracts from pigs given exogenous 1,25-(OH)2D3.
A renal mitochondrial cytochrome P 450 preparation from pigs treated with exogenous 1,25-(OH)2D3 was reconstituted with an NADPH-generating system, adrenodoxin and adrenodoxin reductase. The reconstituted system catalyzed the conversion of the substrate, 25-OH-D3, to metabolites comigrating with authentic 23,25-(OH)2D3 and 24,25-(OH)2D3 in both straight- and reverse-phase high-performance liquid chromatography systems, which achieve separation of these metabolites from each other as well as from other vitamin D metabolites. The putative 23,25-(OH)2D3 product was resistant to periodate treatment, while the 24,25-(OH)2D3 product was sensitive, providing additional evidence for the identity of the products. Although induction of 24-hydroxylase activity has been studied using renal homogenates from several species, only recently have techniques become available to study the activity of the enzyme in a solubilized and reconstituted form. Using these techniques, the present study shows that production of 24,25-(OH)2D3 was increased more than 80-fold with 1,25-(OH)2D3 treatment compared with untreated controls, an effect much greater than that previously observed with homogenates. In addition, production of both 23,25-(OH)2D3 and 24,25-(OH)2D3 varied with substrate concentration and was consistent with a monooxygenase-linked enzyme reaction.
Gray
R W
RW
Department of Medicine, Medical College of Wisconsin, Milwaukee.
Omdahl
J L
JL
Ghazarian
J G
JG
Horst
R L
RL
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D004790:Enzyme Induction; D005260:Female; D007668:Kidney / Q000187:drug effects* / Q000201:enzymology*; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D012995:Solubility; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000187:drug effects; D013552:Swine; D014807:Vitamin D / Q000302:isolation & purification / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol